The present invention is directed to a method for use of enzymes from hyperthermophilic Archaea in the production of product at the expense of added carbon while recycling pyridine nucleotides. In a preferred method of the invention, the enzymes used are glucose dehydrogenase and alcohol dehydrogenase and the product is an alcohol or aldehyde. In the most preferred embodiment both enzymes are from Sulfolobus solfataricus; glucose dehydrogenase is derived directly from S. solfataricus, and the alcohol dehydrogenase is in a recombinant form. Using the method of invention, generation of alcohol from carbohydrate can be accomplished at elevated temperatures which simplify the recovery (facilitate the removal) of alcohol by volatilization, thereby driving the reaction toward production of additional alcohol.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The production of many amino acids such as L-alanine, leucine and glutamate have been accomplished using enzymes that regenerate pyridine nucleotides (Hasumi, et al., 1995; Hasumi, et al., 1996; Itozawa, et al., 1995). In some cases, pyridine nucleotides have been regenerated with the bioreduction of carbonyl compounds (Itoh, et al., 1992; Itozawa, et al., 1995). The regeneration methods used include electricity (Fassouane, et al., 1990) and enzymes (Woodward, et al., 1996). Enzymes which have been used in the regeneration of pyridine nucleotides include glucose dehydrogenase from Thermoplasma acidophilum (Woodward, et al., 1996) and alcohol dehydrogenase from horse liver (Tsuji, et al., 1994).
Current efforts to improve biofuel ethanol production focus on fermentation technology. Large reactor volumes in the conventional production methods are required however to achieve dilute ethanol concentrations needed to overcome microbial ethanol sensitivity. Fermentative synthesis therefore typically continues until the concentration of product is toxic to the microbial population; synthesis is then complete and a separate process is undertaken to recover product. This creates additional constraints on ethanol recovery.
Enzymatic approaches to ethanol synthesis preferably utilize enzymes that have broad substrate specificity, accept several cofactors and which are active and stable at the room temperatures typically employed. Enzymes can be inactivated in a number of ways, such as denaturation (heat or solvent), oxidation and dissociation. Additional considerations are necessary when the enzyme used for conversion of substrate to desired product is reversible and/or affected by product accumulation. Current efforts to address inactivation by product accumulation include monitoring progress of the reaction by measuring substrate concentration (Wong et al., 1985) and other reaction parameters such as temperature, rate and cofactor concentrations. The duration of the synthesis process is therefore limited because the reaction must be stopped before accumulated product inactivates the enzymes. The choice of recovery method depends upon the product to be recovered, for example, recovery of product may be by chemical extraction (Wong et al., 1985) or it may involve semipermeable membranes.
Because industrial synthesis cannot typically proceed under the conditions necessary for recovery of product, recovery is undertaken after synthesis is complete. Thus, it is desirable to establish new methodologies, which simplify the process of synthesis and increase recovery of product and which are capable of overcoming microbial sensitivity to product formation. The present invention solves this need as illustrated herein.
The present invention is directed to a method for use of enzymes from hyperthermophilic Archaea in the production of product at the expense of added carbon while recycling pyridine nucleotides. In a preferred method of the invention, the enzymes used are glucose dehydrogenase and alcohol dehydrogenase and the product is an alcohol or aldehyde. In the most preferred embodiment, both enzymes are from Solfolobus solfataricus; glucose dehydrogenase is derived directly from S. solfataricus, and the alcohol dehydrogenase is in a recombinant form. Using the method of invention, generation of alcohol from carbohydrate can be accomplished at elevated temperatures which simplify the recovery (facilitate the removal) of alcohol by volatilization, thereby driving the reaction toward production of additional alcohol.